High-risk human papillomaviruses (HPV) are the causative agents of cervical cancer. The life cycle of HPV Is tied to the differentiation status of its host cell, the keratinocyte. Differentiation triggers the productive phase of the life cycle, which includes viral genome amplification, late gene expression and virion production. In contrast to normal cells, which exit the cell cycle upon differentiation, expression of the HPV E6 and E7 proteins maintain a subset of cells active in the cell cycle largely through degradation of p53 and pRb, respectively The long-term goal of this project is to understand the mechanisms that regulate the activation of differentiation-dependent viral events. Toward this end, I have demonstrated that HPV31 stimulates a low level of caspase activation upon differentiation that is characteristic of the mitochondrial apoptotic pathway and necessary for amplification of viral genomes through cleavage of the viral replication protein E1. In addition, I have recently found that HPV induces an ATM-dependent DNA damage response in differentiating cells, which is necessary for efficient viral genome amplification, as well as caspase activation through the ATM target Chk2. Based on the finding that caspase activation, as well as Chk2 activation, can be stimulated by both E6 and E7, and the identification of caspase cleavage sites in several HPV proteins, I hypothesize that the HPV proteins E6 and E7 induce caspase activation through a DNA damage response to activate late viral events. Specific aims to test this hypothesis are: 1) Identify the mechanism(s) by which HPV activates caspases in differentiating cells by examining the effect of E6 and E7 on the DNA damage response and alteration of mitochondrial membrane integrity, as well as by performing a mutational analysis of E6 and E7 to identify domains that are necessary for Chk2 activation and caspase activation;2) Determine how caspase cleavage of HPV proteins contributes to differentiation-dependent events in the life cycle by using in vitro and in vivo studies. Delineating the pathway by which E6 and E7 mediate activation of a DNA damage response to lead to caspase activation will offer insight as to how HPV modulates apoptotic machinery to facilitate not only the viral life cycle, but possibly transformation as well.